Retrovirus generation and BM transplantation were performed as previously described51 (link),52 (link). Briefly, a total of 50 male BALB/c mice aged 6–8 weeks were used in retroviral transduction and BM transplantation. BM cells were isolated from the donor mice pretreated with 5-FU (250 mg/kg) and infected with retroviruses containing MigR1-BCR/ABL once daily for 2 days in transplant medium. In all, 5 × 105 cells per mice were transplanted into the irradiated (3.4 Gy twice at a 3-h interval) recipient mice through tail vein injection randomly. After 3 weeks of transplantation, BM cells were subjected to morphological examination, flow cytometry analysis, and scRNA-seq analysis. For morphological examination, cells were centrifuged onto a glass slide and subjected to Wright’s staining (Sigma-Aldrich). Light microscopy images were obtained using the Nikko ECLIPSE TS100.
Wright s staining
Manufactured by Merck Group
Sourced in Germany
Wright's staining is a laboratory technique used for the differential staining of blood cells. It is a widely used method for the identification and morphological analysis of various blood cell types, including red blood cells, white blood cells, and platelets. Wright's stain utilizes a combination of eosin and methylene blue dyes to provide a characteristic color pattern that helps distinguish different cellular components and their structural details under a microscope.
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7 protocols using wright s staining
1
Retroviral Transduction and Bone Marrow Transplantation
2
Lung Lavage and Neutrophil Quantification
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Following anesthetization of the rats, the lungs were lavaged with 1 ml ice-cold PBS three times. The number of total cells and neutrophils in the BALF was calculated using Wright's staining (Sigma-Aldrich). The cells in the BALF were collected by centrifugation at 2,500 × g, stained with Wright's stain according the manufacturer's instructions and then neutrophils that were dyed pale purple were counted using a cell counting plate.
3
Quantifying Megakaryocytes in Bone Marrow
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The number of megakaryocytes in a 1.5×3.5-cm area of a standard bone marrow smear were determined under microscope (BX53; Olympus Corporation, Tokyo, Japan) using Wright's staining (Sigma-Aldrich, St. Louis, MO, USA) in all patients and donors, as described previously (17 (link),18 (link)). Megakaryocyte morphology was also analyzed in all patients and donors (17 (link),18 (link)).
4
Bladder Cancer Cell Chemotaxis and Invasion
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Chemotaxis of bladder cells to 10% FBS in MCoy’s medium was evaluated using a modified Boyden’s chamber with 8 μm pore polycarbonate membrane inserts (Transwell; Corning Life Sciences, PZ HTL SA, Warsaw, Poland), and the chemotactic cells were visualized with Wright’s staining (Sigma-Aldrich, Darmstadt, Germany), as previously described [64 (link)]. BSA MCoy’s medium 0.5% was used as a negative control. Before the experiment, RT4 and T24 cells were treated with sorafenib at IC50 concentration or CR-BAY aggregates for 24 h. 2.0 × 104 RT4 and T24 cells in 0.5 mL in starving medium 0.5% BSA MCoy’s with or without the inhibitor were seeded per one insert. Bladder cancer cells’ invasion ability was measured in Matrigel-coated Transwell insert chambers (Corning LifeSciences, PZ HTL SA, Warsaw, Poland). The inserts were coated with 1 mg/mL Matrigel matrix according to the manufacturer’s recommendations. Methods used in the cell invasion assay were similar to those in the cell chemotactic assay.
5
Puberty Onset Measurement in Mice
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C57 and C3H mice (Additional file 5 : Fig. S5a) were respectively selected to measure puberty onset. Vaginal opening and the first vaginal cornification were obtained and evaluated as previously described [37 (link), 50 (link), 51 (link)]. The mice were daily examined from 17 dpp until the date of vaginal opening (Additional file 5 : Fig. S5b). Daily vaginal smear was performed from the day of vaginal opening till the first emerging day of vaginal epithelial cornification, which was defined as the first vaginal cornification (Additional file 5 : Fig. S5c). Vaginal smears were stained by Wright’s staining (Sigma-Aldrich) and evaluated as previous description [31 (link)].
6
MDSC Subsets Identification Workflow
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The MDSCs populations were sorted using a FACSAria II, according to the following criteria: PMN-MDSCs as LIN−HLA-DRlow/−CD33+CD66b+CD14−, Mo-MDSCs as LIN−HLA-DRlow/−CD33+CD66b−CD14+, and e-MDSCs as LIN−HLA-DRlow/−CD33+CD66b−CD14−, followed by cytospin preparations (Cytospin, Sheldon, UK) and the Wright's staining (Merck, Darmstadt, Germany). Slides were analyzed by Olympus XC50 camera (Olympus, Tokyo, Japan).
7
Colony-forming Ability Evaluation
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The colony-forming assay was performed to assess the colony-forming ability of the cells. Briefly, the CD34+ cells from the initial UCB samples or the cultured cells were incubated in methylcellulose medium with recombinant cytokines (MethoCult GF+ H4435; StemCell Technologies Inc.) at 2×103 in 35-mm tissue culture dishes (Costar, Lowell, MA, USA). The dishes were incubated at 37°C in a humidified atmosphere with 50 ml/l CO2 in air. All cultures were carried out in triplicate. After 14 days of culture, colonies belonging to burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte/macrophage (CFU-GM), colony-forming unit-macrophage (CFU-M) and colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) consisting of ≥50 cells were scored under an inverted microscope (Olympus IX81, Olympus). To assess the accuracy of in situ identification, individual colonies were selected and stained with Wright's staining (Merck, Darmstadt, Germany) for the morphological identification of cells.
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